1. Field of the Invention
The present invention relates to the use of plasminogen activator as an anti-inflammatory agent.
2. Description of the Related Art
Plasminogen activators play an important physiological role in the regulation of thrombolysis. This action is exploited therapeutically to promote reperfusion in conditions such as, for example, acute myocardial infarction, pulmonary embolism, and thrombotic stroke.
An equilibrium between two opposing reactions, coagulation which forms blood clots and fibrinolysis which dissolves blood clots, maintains a patent and intact vascular endothelium. To stop blood loss from a leaking blood vessel, blood clots form a hemostatic plug at the site of a break in the vessel wall. But if the blood clot obstructs flow through a blood vessel, the result may be, for example, a myocardial infarction, a pulmonary embolism, or a thrombotic stroke.
The interruption of flow through the blood vessel will lead to tissue ischemia. In this condition, the tissue is deprived of oxygen and becomes jeopardized, a state in which the tissue is injured but still potentially viable. If however the hypoxic condition is maintained for a period of several hours, the tissue becomes necrotic and cannot recover. It is therefore important that reperfusion, the restoration of blood flow, be accomplished as soon as possible to minimize tissue necrosis. However, even if reperfusion is accomplished before tissue necrosis occurs, leukocytes may become activated and infiltrate the jeopardized tissue. Consequently, reperfusion only leads to partial recovery of jeopardized tissue, the remainder being permanently damaged by leukocyte mediated oxidative injury or other pathophysiological mechanisms (Hansen, Circulation 91:1872-1885, 1995).
In particular, patients with acute myocardial infarction have significantly reduced mortality when treated with a plasminogen activator. This benefit is due to blood clot fibrinolysis and timely opening of the infarct-related artery. However, once the infarct-related artery is patent, neutrophils can contribute to the reperfusion injury that accompanies reversal of ischemia. Reperfusion of the myocardium is associated with neutrophil activation and infiltration. The nature of the neutrophil-mediated injury is not fully characterized but is in part due to the production of superoxide anion (O2xe2x88x92) and/or related oxidative products. This principle (activation of white blood cells release of toxic mediators and resultant pathophysiology in the host) is common to many inflammatory diseases including, but not limited to, acute respiratory distress syndrome, cystic fibrosis, asthma, arthritis, and nephritis.
A drawback of thrombolytic therapy with plasminogen activator is the increased incidence of stroke and intracerebral hemorrhage in treated patients. The generation of fibrin fragments and depletion of fibrinogen caused by the exogenous plasminogen activator is a direct cause of excessive bleeding disorders.
Side-effects of existing anti-inflammatory agents such as steroidal (e.g., corticosteroids) and non-steroidal (e.g., aspirin, acetaminophen, ibuprofen) drugs include growth retardation, osteoperosis, gastric and renal toxicity, and adrenal suppression. In addition, these agents can impair the healing process due to inhibition of neutrophil infiltration by steroidal and non-steroidal drugs. Most non-steroidal drugs are also limited to oral formulations.
We have now found that plasminogen activator can reduce tissue injury by acting as an anti-inflammatory agent. More generally, plasminogen activator reduces tissue damage due to leukocyte mediated oxidative injury. Plasminogen activator inhibits leukocyte generation of oxygen radicals (e.g., hydroxides, peroxides, superoxides) by a mechanism that is independent of thrombolytic activity and scavenging of oxygen free radicals. By separating the thrombolytic and the anti-inflammatory functions of plasminogen activator, the present invention provides a method of reducing tissue damage due to oxidative injury (e.g., reperfusion injury) while mitigating complications from excessive bleeding, such as stroke and intracerebral hemorrhage. Moreover, because the present invention does not inhibit neutrophil migration and infiltration, use of plasminogen activator as an anti-inflammatory agent will not interfere with processes mediated at least in part by neutrophils such as, for example, wound healing or tissue remodeling, which is a shortcoming of existing steroidal and non-steroidal anti-inflammatory agents.
It is an object of the invention to reduce cell and/or tissue damage due to oxidative injury.
It is a further object of the invention to inhibit oxidant production by leukocytes.
Another object of the invention is to use plasminogen activator as an anti-inflammatory agent.
Yet another object of the invention is to treat a human or animal afflicted with an inflammatory disease.
It is an object of the invention to select and/or develop derivatives of plasminogen activator that retain the anti-inflammatory property of plasminogen activator.
Another object of the invention is to provide a derivative of plasminogen activator that can be used as an anti-inflammatory agent.
Yet another object of the invention is to provide a non-thrombolytic form of plasminogen activator useful as an anti-inflammatory agent.
In one embodiment of the invention, a plasminogen activator or derivative thereof is administered to an organism, and thereby reduces oxidative injury to tissue of the organism. Preferably, the organism is afflicted with an autoimmune disease or an inflammatory disease. The organism may be a human or an animal. The oxidative injury may be mediated by an inflammatory cell (e.g., neutrophil, macrophage, monocyte, eosinophil, mast cell, basophil). Tissue at risk of oxidative injury may include, but is not limited to, any blood-prefused tissue (e.g., myocardiun, lung, brain) or any bone or joint.
In a second embodiment of the invention, a plasminogen activator or derivative thereof is applied to an inflammatory cell (e.g., neutrophil, macrophage, monocyte, eosinophil, mast cell, basophil), and thereby reduces oxidant production by the inflammatory cell.
A third embodiment of the invention is the treatment of an organism afflicted with an inflammatory disease by administering a plasminogen activator or derivative thereof, and thereby reducing or alleviating a symptom of the disease caused by inflammation. The organism may be a human or an animal. Preferably, the organism is afflicted with an acute or chronic inflammatory disease.
A fourth embodiment of the invention is the prophylactic treatment of an organism at risk for development of an inflammatory illness by administering a plasminogen activator or derivative thereof, and thereby preventing onset of the illness or reducing the severity of a symptom of the illness. The organism may be a human or an animal. Preferably, the organism is at risk for development of an acute or chronic inflammatory illness.
For the above embodiments of the invention, plasminogen activator may be tissue-plasminogen activator (tPA), urokinase (uPA), reteplase (rPA), or a derivative thereof. Such plasminogen activators may be termed xe2x80x9cendogenous plasminogen activatorsxe2x80x9d to distinguish them from streptokinase. Tissue plasminogen activator and urokinase are xe2x80x9cmammalian plasminogen activatorsxe2x80x9d whereas streptokinase is produced by bacteria. For these two definitions, no distinction is made between native protein and recombinantly produced protein (e.g., non-glycosylated). Instead, xe2x80x9cendogenousxe2x80x9d may mean derived from the species of the organism or the inflammatory cell being treated and xe2x80x9cmammalianxe2x80x9d indicates the gene encoding the plasminogen activator is derived from a mammal.
Preferred derivatives of endogenous plasminogen activator have the property of reduced fibrinolytic activity, binding a receptor for plasminogen activator, inhibiting oxidant production, inhibiting leukocyte activation, or a combination thereof. More preferably, the derivative of endogenous plasminogen activator binds urokinase receptor with increased affinity, reduces production of superoxide anion by an inflammatory cell (e.g., neutrophil, macrophage, monocyte, eosinophil, mast cell, basophil), reduces release of other mediators of inflammation (e.g., arachidonate metabolites, cytokines, histamine, monokines, nitric oxide, proteases, serotonin) by an inflammatory cell or the endothelium, or a combination thereof. The 15 Kd amino-terminal fragment (ATF) (amino acid residues 1-135) of urokinase is an example of such a derivative, binding the urokinase receptor but lacking fibrinolytic activity.
A fifth embodiment of the invention is to provide structural variants of an endogenous plasminogen activator, to screen the structural variants for their ability to reduce inflammation, and to select those structural variants which reduce inflammation. The structural variant may be produced recombinantly, by peptide synthesis, by protease cleavage, or by chemical modification. Preferred structural variants have the property of reduced fibrinolytic activity, binding a receptor for plasminogen activator, inhibiting oxidant production, inhibiting leukocyte activation, or a combination thereof. The 15 Kd amino-terminal fragment (ATF) (amino acid residues 1-135) of urokinase is an example of such a preferred structural variant, binding the urokinase receptor but lacking fibrinolytic activity. More preferably, the structural variant binds the receptor for plasminogen activator with increased affinity, reduces production of oxidant radicals and/or another marker of leukocyte activation, reduces cellular degranulation, reduces vascular permeability, or a combination thereof.
Endogenous plasminogen activator may be tissue-plasminogen activator (tPA), urokinase (uPA), or a derivative thereof. A plasminogen derivative with reduced fibrinolytic activity is preferred, but not necessary. A plasminogen activator derivative that binds the plasminogen activator receptor on leukocytes, thereby inhibiting production of oxidants and/or other markers of leukocyte activation, is also preferred but not necessary.
The advantages of the invention include the ability to inhibit inflammation and tissue injury using a plasminogen activator that does not cause excessive bleeding, and provision of a novel class of anti-inflammatory agents that reduces tissue injury caused by leukocyte production of oxidants and proteases, without interfering with other leukocyte functions such as, for example, migration and infiltration.